Summary of work: GADD153 is a highly conserved mammalian gene whose expression is increased in response to a variety of stresses. It has been implicated in both growth arrest and apoptosis, but its functional role in these processes is far from clear. GADD153 is a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors. It cannot bind to consensus C/EBP binding sites, but can act as a negative regulator of these C/EBPs by virtue of its ability to dimerize with them and inhibit their binding to DNA. It has also recently been suggested that GADD153-C/EBPbeta dimers can act as positive regulators of transcription through interaction with novel sites. Studies in this project have focused on the regulation of GADD153 gene expression and the function of the GADD153 protein during the cellular response to diverse stimuli. With respect to regulation, recent efforts have concentrated on the role of the CREB-family transcription factor ATF6 (implicated in regulating gene expression during the response to stress to the endoplasmic reticulum (ER stress)), in regulating GADD153 transcription. We have provided evidence that overexpression of ATF6 transactivates the GADD153 promoter and that antisense ATF6 attenutates GADD153 expression by ER stress. Current studies are aimed at further characterizing the role of ATF6 in regulating GADD153 expression by other stresses and identifying the ATF6-responsive site in the GADD153 promoter. Studies on the function of GADD153 have focused on its ability to influence cell survival during stress. Employing model systems in which GADD153 protein is either constitutively expressed at high levels or placed under inducible expression we have found that GADD153 overexpression heightens the cells sensitivity to a variety of stressful treatments resulting in apoptosis. GADD153 overexpression also leads to a reduction in cellular glutathione levels, elevated amounts of reactive oxygen species and reduced levels of Bcl-2 protein. Current studies are further exploring the association between these phenomena and the reduced survival of GADD153-expressing cells. We are also investigating the molecular mechanism whereby GADD153 represses Bcl-2 expression. Our findings suggest that GADD153 serves a pro- apoptotic function through perturbations in cellular redox status. - GADD153, ATF3, stress response, transcriptional regulation, C/EBP, apoptosis, Bc1-2.